CN116396932B - Blood cell pretreatment composition and application thereof - Google Patents
Blood cell pretreatment composition and application thereof Download PDFInfo
- Publication number
- CN116396932B CN116396932B CN202310664029.9A CN202310664029A CN116396932B CN 116396932 B CN116396932 B CN 116396932B CN 202310664029 A CN202310664029 A CN 202310664029A CN 116396932 B CN116396932 B CN 116396932B
- Authority
- CN
- China
- Prior art keywords
- pretreatment composition
- kit
- pretreatment
- mass concentration
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 27
- 210000000601 blood cell Anatomy 0.000 title claims abstract description 18
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 18
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims abstract description 14
- 102000004142 Trypsin Human genes 0.000 claims abstract description 13
- 108090000631 Trypsin Proteins 0.000 claims abstract description 13
- 239000012588 trypsin Substances 0.000 claims abstract description 13
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 12
- 239000012091 fetal bovine serum Substances 0.000 claims abstract description 12
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229930040373 Paraformaldehyde Natural products 0.000 claims abstract description 6
- 229920002866 paraformaldehyde Polymers 0.000 claims abstract description 6
- 239000011780 sodium chloride Substances 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 13
- 238000004458 analytical method Methods 0.000 claims description 10
- 238000010186 staining Methods 0.000 claims description 9
- 210000003743 erythrocyte Anatomy 0.000 claims description 6
- 238000002955 isolation Methods 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000002981 blocking agent Substances 0.000 claims description 2
- 239000013592 cell lysate Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000003381 stabilizer Substances 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims 2
- 230000003712 anti-aging effect Effects 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 12
- 239000008363 phosphate buffer Substances 0.000 abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 43
- 239000000523 sample Substances 0.000 description 29
- 210000004369 blood Anatomy 0.000 description 14
- 239000008280 blood Substances 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 14
- 239000000872 buffer Substances 0.000 description 13
- 238000007710 freezing Methods 0.000 description 9
- 230000008014 freezing Effects 0.000 description 9
- 239000011886 peripheral blood Substances 0.000 description 8
- 210000005259 peripheral blood Anatomy 0.000 description 8
- 230000000087 stabilizing effect Effects 0.000 description 8
- 238000005406 washing Methods 0.000 description 7
- 239000006285 cell suspension Substances 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 238000013112 stability test Methods 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 5
- 239000000834 fixative Substances 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 101000831007 Homo sapiens T-cell immunoreceptor with Ig and ITIM domains Proteins 0.000 description 4
- 102100024834 T-cell immunoreceptor with Ig and ITIM domains Human genes 0.000 description 4
- 210000002865 immune cell Anatomy 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 102100027581 Forkhead box protein P3 Human genes 0.000 description 3
- 101000861452 Homo sapiens Forkhead box protein P3 Proteins 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- 239000012103 Alexa Fluor 488 Substances 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000012758 nuclear staining Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000013062 quality control Sample Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- ZMJPCIAEJKVKMQ-UHFFFAOYSA-M [4-[[4-[benzyl(methyl)amino]phenyl]-[4-(dimethylamino)phenyl]methylidene]cyclohexa-2,5-dien-1-ylidene]-dimethylazanium;chloride Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC(=CC=1)N(C)CC=1C=CC=CC=1)=C1C=CC(=[N+](C)C)C=C1 ZMJPCIAEJKVKMQ-UHFFFAOYSA-M 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000004159 blood analysis Methods 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012520 frozen sample Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the field of biological detection, in particular to a pretreatment composition of blood cells and application thereof. The present invention provides a pretreatment composition for blood cells, comprising: paraformaldehyde, glutaraldehyde, ethylene glycol, sodium chloride, phosphate buffer, trypsin, and fetal bovine serum. The pretreatment composition provided by the invention is simple and convenient to operate, does not need special instruments and consumables, does not relate to PBMC separation operation, originally needs 2-3 hours for separation operation, and can be finished in 10 minutes. More effective clinical data can be obtained.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a pretreatment composition of blood cells and application thereof.
Background
Flow cytometry is a very mature high-tech technology for rapid quantitative analysis and sorting of single cells, has wide application and plays a very important role in both preclinical and clinical processes. Analysis of immune cell subpopulations in body fluid and positioning detection in tissues are taken as an important index for judging cell immune functions, and have important significance on aspects of diagnosing malignant tumors, autoimmune diseases, blood system diseases, organ transplantation of the same kind of tissues, diagnosis of various infectious diseases, research of pathogenesis, guiding immunotherapy, judging prognosis and the like. With the advent of anti-T cell monoclonal antibodies and the establishment of various detection methods. Immune cell subpopulation detection has found wide application in basic medicine and clinical medicine research.
For the study of immune cell subpopulations, the most widely used study sample is whole blood, since it does not require additional handling and maintains the sample in a more original state. Preclinical animal testing is a batch operation, and the testing laboratory is typically located in close proximity to the animal facility for ease of testing, and thus, more is available for whole blood analysis. However, in clinical practice, since clinical research centers are generally far away from analytical laboratories and even distributed across the country, whole blood samples place high demands on the timeliness of analysis, and at the same time, since the number of samples analyzed per analysis batch is small, the analysis cost of a single sample is high, and the waste of experimental resources is also caused. In order to more conveniently simulate the external blood immune environment, the most common practice is to separate peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cell, abbreviated as PBMC), remove polynuclear cells and erythrocytes, and obtain mononuclear cells by using a solution (called stratified solution) between specific gravity of the polynuclear cells and erythrocytes for density gradient centrifugation. For most targets, isolated PBMC are becoming increasingly popular because they can be stored for longer periods of time at-80℃or in liquid nitrogen.
Limited to instrument configuration (horizontal rotary centrifuges), personnel configuration (personnel with a certain experience are required), aging, etc., the isolation and cryopreservation of PBMCs becomes a major challenge, and not all clinical centers have the ability to perform PBMC isolation and cryopreservation operations. Therefore, there is a need in the art for a blood pretreatment composition that does not require PBMC isolation procedures, is simple to operate, does not require special instrumentation and consumables, and still ensures the accuracy of subsequent results.
Disclosure of Invention
In view of this, it is an object of the present invention to provide a pretreatment composition for blood cells, comprising: paraformaldehyde, glutaraldehyde, ethylene glycol, sodium chloride, phosphate buffer, trypsin, and fetal bovine serum.
The pretreatment composition provided by the invention is simple and convenient to operate, does not need special instruments and consumables, does not relate to PBMC separation operation, originally needs 2-3 hours for separation operation, and can be finished in 10 minutes. More effective clinical data can be obtained. Many projects, limited by the age of transportation of samples and lengthy PBMC isolation procedures, may involve clinical studies in which 100 subjects may only be available for the analytical detection of an effective subpopulation of immune cells in 40 subjects or less.
In some specific embodiments, the concentration of paraformaldehyde is 0.2-0.7% by mass, glutaraldehyde is 0.2-0.8% by mass, ethylene glycol is 0.01-0.04% by mass, sodium chloride is 0.05-0.5% by mass, phosphate buffer is 0.01-0.06M by mass, trypsin is 0.1% -0.3% by mass, and fetal bovine serum is 5-13% by mass.
In some specific embodiments, the concentration of trypsin is 0.2% by mass and the concentration of fetal bovine serum is 9% by mass.
In a second aspect, the present invention provides a blood cell pretreatment kit comprising a pretreatment composition as described above.
Further, the kit may further comprise other reagents, in particular antibodies, blocking agents, staining agents, etc.
Further, the kit may further comprise a red blood cell lysate.
In a third aspect, the present invention provides a use of the pretreatment composition described above for the preparation of a blood cell pretreatment kit.
In some embodiments, the invention provides a use of the pretreatment composition described above for preparing a blood cell stabilizer.
In a fourth aspect, the present invention provides a method for pretreatment of blood cells, comprising:
1) Pretreating a sample with a pretreatment composition as described above;
2) Without isolation of PBMCs; and
3) A subsequent analysis step is performed.
Drawings
FIG. 1 is a graph showing the results of stability tests without trypsin and fetal bovine serum;
FIG. 2 is a graph showing the results of the stability test with trypsin and fetal bovine serum.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Example 1 blood cell pretreatment method
Fresh human peripheral blood and blood cell pretreatment mixture (0.5% of paraformaldehyde, 0.5% of glutaraldehyde, 0.02% of ethylene glycol, 0.2% of sodium chloride, 0.05M of phosphate buffer, 0.2% of trypsin and 9% of fetal bovine serum) are mixed according to a volume ratio of 5:1, and the mixture is left stand for about 2 hours at 2-8 ℃ after uniform mixing.
Example 2 precision test
Reagent: PE anti-human CD3 (Biolegend, B314617), perCP/cyanine5.5 anti-human CD4 (Biolegend, B361883), brilliant Violet 785TM anti-human CD8a (Biolegend, B340251), ki-67 Monoclonal Antibody (SolA 15) eFluor 450 (Invitrogen, 2312457), alexa Fluor 488 anti-human FOXP3 (Biolegend, B358132), PEanti-human CD3 (Biolegend, B326669), alexa Fluor 488 anti-human FOXP3 (Biolegend, B315166), AF647-Tigit isotype control antibody (Jingham organism), AF647-Tigit detection antibody (Jingham organism), mouse serum powder (Jackson ImmunoResearch, 015-000-120), sizing buffer (Biolegend, 420201), 1×pbs (Corning, 21-040-CVR), 0.4% trypan blue dye (Invitrogen, T10282), AO/PI dye (Count star, RE 010212), 10× RBC Lysing Buffer (BD, 555899), e bioscience FOXP3/Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00), eBioscience Flow Cytometry Staining Buffer (Invitrogen, 00-4222-26), 1640 medium (Sigma, R1145-500 ML), fetal Bovine Serum (Gibco, 10099-141), penicillin/streptomycin (Penstrep) (Gibco, 15140), glutaMAX (100×) (Gibco, 35050-061), PHA (Sigma, L8754), cryos CS10 (Stem Cell Technology, 07930)
The experimental process comprises the following steps:
preparing a quality control sample:
a) Cell resuscitation: the cryopreserved stimulated PBMC were removed according to the experimental plan, the cells were rapidly thawed in a 37℃water bath, immediately after the last ice was thawed, the cells were transferred to a pre-warmed stabilizing buffer (recommended volume 10 mL), mixed well, centrifuged at 500 Xg for 5 mins at room temperature, and the cell supernatant was discarded.
b) Resuspension count: cells were resuspended with an appropriate volume of stabilizing buffer. Cell concentration and viability were determined using a cytometer. After centrifugation at 500 Xg for 5 mins at room temperature, the cell supernatant was discarded.
c) And (3) fixedly storing: stimulated PBMC cells were resuspended to about 5X 10 with PHA (about 10 ug/mL) in 1640 complete medium 6 cell/mL (cell density can be adjusted according to the requirement), adding whole blood fixative, standing at 2-8deg.C for 2-4 h (same preservation time as clinical sample), freezing and preserving according to the requirement, and using fresh sample in the time range as quality control or frozen sample at-70deg.C as quality control (consistent with clinical sample requirement).
d) For quality control samples to be frozen and stored: and (3) distributing proper volume according to the requirement, adding the cell freezing solution (Crosotor CS 10) with the same volume, subpackaging the proper volume into a freezing tube, storing the freezing tube into a program cooling box, and freezing in a refrigerator at the temperature of-70 ℃. Samples that were frozen for at least 4 hours were available for detection.
Note that: the ratio of the volume of the cell suspension to the whole blood fixative is referred to the ratio of the whole blood fixation, so that the concentration of each active ingredient is ensured to be suitable.
Sample pretreatment:
for fresh quality control samples:
after the sample is mixed evenly, the cell suspension is taken out, the cell suspension is transferred to a proper volume of stabilizing buffer for washing, and after 500 Xg centrifugation is carried out for 5 minutes at room temperature, the cell supernatant is discarded.
For fresh human peripheral blood samples:
(1) Split red: adding 1×erythrocyte lysate with the volume 9 times that of the sample, mixing, and incubating at room temperature in dark place for 15 min. After centrifugation at 500 Xg for 5 mins at room temperature, the supernatant was discarded.
(2) Resuspension count: cells were resuspended with an appropriate volume of stabilizing buffer. Cell concentration and viability were determined using a cytometer. After centrifugation at 500 Xg for 5 mins at room temperature, the cell supernatant was discarded.
The experimental steps are as follows:
(1) Cell plating: the cells were resuspended to approximately 2.5X106 cells/mL with a stabilizing buffer after pretreatment of the sample. Split-loading into 96-well V-bottom plates, 200 μl (approximately 5×105 cells per well, if the cell volume is insufficient, resuspended in volume as needed), centrifuging 500×g at room temperature for 5 min, and removing supernatant.
Each clinical sample is split into two tubes (including a detection tube and a control tube), and the rest samples are split into separate samples according to the requirement.
(2) Blocking incubation 1: adding surface dyeing sealing solution 1, 50 μl/hole, mixing, and incubating at room temperature for 15 min + -3 min
(3) Surface staining antibody incubation: adding the surface staining antibody mixture, 50 mu L/hole, mixing, and incubating for 15 min+/-3 min at room temperature.
(4) 2 times of washing: 100 mu L staining buffer or 1 XPBS was added to each well, centrifuged at 500 Xg for 5 min at room temperature, the supernatant was discarded, washing was repeated once with 200 mu L staining buffer or 1 XPBS, and the mixture was homogenized, centrifuged at 500 Xg for 5 min at room temperature, and the supernatant was discarded.
(5) Fixing and rupture of membranes: vortex cell, add 200 μl Foxp3 fixation/rupture working fluid into each well, mix well, incubate for 30 min±5 min at room temperature in the dark. Centrifuge at 500 Xg for 5 min at room temperature and discard the supernatant.
(6) 2 times of washing: 200. Mu.L of 1 Xmembrane rupture liquid was added to each well, mixed well, centrifuged at 500 Xg for 5 min at room temperature, and the supernatant was discarded. The washing procedure was repeated once.
(7) Blocking and incubation 2: adding nuclear stain blocking solution 2, 50 mu L/hole, mixing, and incubating at room temperature in dark place for 15 min + -3 min
(8) Nuclear staining antibody incubation: 50 mu L of the mixed solution of the nuclear staining antibodies is added into each hole, and the mixture is uniformly mixed and incubated for 30 min plus or minus 5 min at room temperature in a dark place.
(9) 2 times of washing: 100. Mu.L of 1 Xmembrane rupture liquid was added to each well, and the mixture was centrifuged at 500 Xg for 5 min at room temperature, and the supernatant was discarded. 200. Mu.L of 1 Xmembrane rupture liquid was added to each well, mixed well, centrifuged at 500 Xg for 5 min at room temperature, and the supernatant was discarded.
(10) Sample resuspension: 300 [ mu ] L staining buffer resuspended cells were added per well and the cell suspension was transferred to a 5 mL flow tube if desired. The sample can be collected immediately, or can be stored at 2-8deg.C in dark place, and can be collected in 24 h.
(11) Sample collection: sample collection was performed using Beckman Coulter CytExpert v 2.4.2.4, and samples were homogenized at low speed using a vortex apparatus to form a homogeneous single cell suspension prior to flow meter collection, and samples were collected until at least 40,000 events (cell number) were harvested in the "cd3+" gate. The sample collection speed is not more than 10000 events/second, and the sample collection speed can be adjusted to be proper by adjusting the sampling speed, the threshold value or adding stabilizing. If the number of the sample cells is insufficient, the sample cells are collected as much as possible on the premise of avoiding the suction of the instrument.
(12) And (3) data preservation: and after the sample collection is completed, the experimental file and the FCS file are timely derived and directly stored in a folder appointed by the security disk. Data analysis was performed using FlowJo v10 software. The data may be further processed using Microsoft Excel 2016, where the "set precision to precision displayed" option will be checked when computing in Microsoft Excel.
Note 1: the 5 mL flow tube can be selected to be used for experiments according to the requirement, the washing volume is required to be enlarged to 2 mL, and the rest is not required to be adjusted.
And (2) injection: the cell is resuspended and uniformly mixed, and shake for 2 min at 1300 rpm at room temperature by using a plate shaking machine or form single cell suspension finally by selecting other suitable modes.
And (3) injection: cells were not treated with fixative and were counted based on viable cell concentration and were treated with fixative and counted based on total cell concentration.
After the peripheral blood of the healthy person is collected, the blood cell pretreatment composition is added, the blood cell pretreatment composition is preserved for about 2 hours at the temperature of 2-8 ℃, proper volumes are distributed according to the requirement and are placed at the temperature of-70 ℃ for freezing (more than 4 hours) for carrying out the intra-batch and inter-batch precision evaluation of the method, 3 analysis batches are completed by two different analysts on 2 different analysis days, each analysis batch comprises samples prepared from the whole blood of the 3 healthy persons, and each sample detection comprises a DT tube and an FMO tube.
The 3 analysis batch results met: the proportion of CD3+, CD4+, CD8+, TIGIT, ki-67 and FoxP3 positive cells of the samples prepared from the whole blood of the 2/3 healthy people is less than or equal to 35.0 percent of CV which is required to meet the precision in batches and between batches, and the method is precise and has better reproducibility. See tables 1, 2, and 3 for specific results.
TABLE 1
TABLE 2
TABLE 3 Table 3
Note that: in the table, "Intra" indicates "precision in lot"; "Inter" means "precision between lots"; bold and italic indicate that the acceptance criteria are not met.
Example 3 stability test at-70℃for 1 week after pretreatment
The stability test procedure was essentially the same as in example 1, except that the whole blood cryopreserved stability sample needed to be resuscitated prior to drug incubation.
(1) And (3) resuscitating a sample: rapidly shaking for resuscitation in a 37 ℃ water bath, immediately after melting the last ice, transferring the sample into a preheated stabilizing buffer (recommended volume 10 mL) with a proper volume, mixing well, centrifuging at 500 Xg for 5 min at room temperature, and discarding the cell supernatant.
(2) Resuspension count: cells were resuspended with an appropriate volume of stabilizing buffer. Cell concentration and viability were determined using a cytometer. After centrifugation at 500 Xg for 5 mins at room temperature, the cell supernatant was discarded.
The stability test needs to include 3 different batches of human peripheral blood, and single well detection includes a DT tube and a FMO tube. Adding fixative after collecting human peripheral blood, preserving at 2-8deg.C for about 2 hr, taking out appropriate volume as baseline detection sample, sampling baseline, distributing appropriate volume according to requirement, packaging appropriate volume (such as 1.8 mL) into freezing tube, and freezing at-70deg.C. The stability of storage at-70℃for 1 week was evaluated by storing at 2-8℃for about 2 hours after baseline sampling.
The stability results after freezing for 1 week meet the following conditions: the ratio of CD3+, CD4+, CD8+, TIGIT, ki-67 and FoxP3 positive cells of the sample prepared from the whole blood of the 2/3 healthy person is compared with the baseline, the percentage of CV is less than or equal to 25.0%, and the method stability is better. See table 4 for specific results.
After the whole blood sample is stored for 2 hours at 2-8 ℃ and is stored for 38H at-20 ℃, the stability test meets the percentage CV less than or equal to 30.0%, and the method stability is better.
TABLE 4 Table 4
Example 4 comparative test of the effect of trypsin and fetal bovine serum on stability
The procedure was the same as in example 2.
Stability samples without and with trypsin and foetal calf serum added were tested.
Stability results without trypsin and foetal calf serum show that in cd3+ T lymphocytes: 2/3 batches of peripheral blood did not meet the acceptance criteria compared to% CV for foxp3+ positive cells versus baseline; cd4+ T lymphocytes: 2/3 batches of peripheral blood did not meet the acceptance criteria compared to% CV for foxp3+ positive cells versus baseline; TIGIT positive cells were compared to% CV at baseline, 2/3 batches of peripheral blood failed to meet the acceptance criteria and failed to meet the acceptance criteria, and the method was less stable. See fig. 1 for specific results.
Stability results with trypsin and fetal bovine serum added showed that in cd3+ T lymphocytes: the ratios of CD3+, CD4+, CD8+, TIGIT, ki-67 and FoxP3 positive cells of the samples prepared from the whole blood of the 2/3 healthy people are compared with the baseline, and the results all meet the preset stability acceptance standard, so that the method has better stability. See fig. 2 for specific results.
Claims (8)
1. A pretreatment composition for blood cells comprising: the anti-aging agent comprises paraformaldehyde, glutaraldehyde, ethylene glycol, sodium chloride, phosphate buffer solution, trypsin and fetal bovine serum, wherein the mass concentration of the paraformaldehyde is 0.5%, the mass concentration of the glutaraldehyde is 0.5%, the mass concentration of the ethylene glycol is 0.02%, the mass concentration of the sodium chloride is 0.2%, the mass concentration of the phosphate buffer solution is 0.05M, the mass concentration of the trypsin is 0.2%, and the mass concentration of the fetal bovine serum is 9%.
2. A pretreatment kit for blood cells comprising the pretreatment composition of claim 1.
3. The kit of claim 2, wherein the kit may further comprise other reagents.
4. A kit according to claim 3, wherein the remaining reagents comprise antibodies, blocking agents, staining agents.
5. The kit of claim 3 or 4, wherein the kit further comprises a red blood cell lysate.
6. Use of the pretreatment composition of claim 1 for preparing a blood cell pretreatment kit.
7. Use of the pretreatment composition of claim 1 for the preparation of a blood cell stabilizer.
8. A method of pretreatment of blood cells, comprising:
1) Pretreating a sample with the pretreatment composition according to claim 1 or the kit according to any one of 2 to 5;
2) Without isolation of PBMCs; and
3) A subsequent analysis step is performed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310664029.9A CN116396932B (en) | 2023-06-07 | 2023-06-07 | Blood cell pretreatment composition and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310664029.9A CN116396932B (en) | 2023-06-07 | 2023-06-07 | Blood cell pretreatment composition and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116396932A CN116396932A (en) | 2023-07-07 |
| CN116396932B true CN116396932B (en) | 2023-08-29 |
Family
ID=87020266
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310664029.9A Active CN116396932B (en) | 2023-06-07 | 2023-06-07 | Blood cell pretreatment composition and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116396932B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0627626A1 (en) * | 1993-06-03 | 1994-12-07 | Burkhardt, Bernd, Dr.med. | Detection of viral or bacterial antigens with flow cytometry |
| US5516695A (en) * | 1993-02-25 | 1996-05-14 | Abbott Laboratories | Multipurpose reagent system for rapid lysis of whole blood |
| WO2000038708A1 (en) * | 1998-12-24 | 2000-07-06 | Phairson Medical Inc. | Treatment and prevention of immune rejection reactions |
| CN105074418A (en) * | 2012-12-28 | 2015-11-18 | 贝克曼考尔特公司 | Compositions for permeabilising fixed blood cells and uses thereof |
| CN113218847A (en) * | 2021-04-12 | 2021-08-06 | 武汉凯普瑞生物技术有限公司 | Hemolytic agent for flow cytometry analysis and preparation method and application method thereof |
| CN113884671A (en) * | 2021-09-27 | 2022-01-04 | 苏州东岭生物技术有限公司 | Flow type dyeing kit and configuration method and application method thereof |
| CN113980814A (en) * | 2021-05-25 | 2022-01-28 | 香港大学深圳医院(深圳市滨海医院) | Composition for rapidly cracking peripheral red blood cells and application thereof |
-
2023
- 2023-06-07 CN CN202310664029.9A patent/CN116396932B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5516695A (en) * | 1993-02-25 | 1996-05-14 | Abbott Laboratories | Multipurpose reagent system for rapid lysis of whole blood |
| EP0627626A1 (en) * | 1993-06-03 | 1994-12-07 | Burkhardt, Bernd, Dr.med. | Detection of viral or bacterial antigens with flow cytometry |
| WO2000038708A1 (en) * | 1998-12-24 | 2000-07-06 | Phairson Medical Inc. | Treatment and prevention of immune rejection reactions |
| CN105074418A (en) * | 2012-12-28 | 2015-11-18 | 贝克曼考尔特公司 | Compositions for permeabilising fixed blood cells and uses thereof |
| CN113218847A (en) * | 2021-04-12 | 2021-08-06 | 武汉凯普瑞生物技术有限公司 | Hemolytic agent for flow cytometry analysis and preparation method and application method thereof |
| CN113980814A (en) * | 2021-05-25 | 2022-01-28 | 香港大学深圳医院(深圳市滨海医院) | Composition for rapidly cracking peripheral red blood cells and application thereof |
| CN113884671A (en) * | 2021-09-27 | 2022-01-04 | 苏州东岭生物技术有限公司 | Flow type dyeing kit and configuration method and application method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116396932A (en) | 2023-07-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dagur et al. | Collection, storage, and preparation of human blood cells | |
| Barnett et al. | Guideline for the flow cytometric enumeration of cd34+ haematopoietic stem cellsprepared by the cd34+ haematopoietic stem cell working party | |
| Corash et al. | Heterogeneity of human whole blood platelet subpopulations. I. Relationship between buoyant density, cell volume, and ultrastructure | |
| EP1021701B1 (en) | Hematology reference control and method of preparation | |
| US7749757B1 (en) | Stabilizing solution for cells and tissues | |
| Kobylka et al. | PRESERVATION OF IMMUNOLOGICAL AND COLONY-FORMING CAPACITIES OF LONG-TERM (15 YEARS) CRYOPRESERVED CORD BLOOD CELLS1 | |
| US20220018745A1 (en) | A method for preparing lymphocyte sample for flow cytometry analysis | |
| US20210389225A1 (en) | A flow cytometric detection method for lymphocyte in immune cells | |
| US20230062518A1 (en) | Stable reference materials for automated hematology testing platforms | |
| Braudeau et al. | An easy and reliable whole blood freezing method for flow cytometry immuno‐phenotyping and functional analyses | |
| Swatler et al. | Isolation and characterization of extracellular vesicles from cell culture conditioned medium for immunological studies | |
| McCoy, Jr | Handling, storage, and preparation of human blood cells | |
| CN116396932B (en) | Blood cell pretreatment composition and application thereof | |
| EP0660664B1 (en) | Preserved, non-infectious control cells for use in the identification of a disease through blood testing | |
| Mezouar et al. | Isolation of human placental mast cells | |
| CN107576789A (en) | A kind of lyophilized NK cell surface antigens quality-control product and preparation method thereof | |
| AU2022436042A1 (en) | Method for detecting a phenotype and a function of a cd141+ dendritic cell subset and kit for use | |
| Fornas et al. | Flow cytometric‐based isolation of nucleated erythroid cells during maturation: An approach to cell surface antigen studies | |
| Madsen et al. | HLA—DR typing of frozen B lymphocytes | |
| Bullock et al. | A novel approach for enzyme histochemical and autoradiographic studies on single cells | |
| FARLEY et al. | An intralaboratory quality control program for quantitation of CD34+ cells by flow cytometry | |
| Jain et al. | The platelet factor‐3 test for detection of canine antiplatelet antibody | |
| D'Souza et al. | A comparison of the choice of monoclonal antibodies for recovery of fetal cells from maternal blood using FACS for noninvasive prenatal diagnosis of hemoglobinopathies | |
| Janetzki et al. | Sample Preparation | |
| CN116724992A (en) | Whole blood frozen stock solution and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |